The present invention relates to methods and kits for characterizing GC rich nucleic acid sequences. More particularly, the present invention relates to methods and kits for amplification, size determination and sequencing of GC rich nucleic acid sequences. Most particularly, the present invention relates to methods and kits for amplification, size determination and sequencing of GC rich nucleic acid sequences, such as the trinucleotide repeats in the FMRI gene causing the Fragile X syndrome, and other genes.
Triple Repeat Mutations
Trinucleotide repeats are the sites of mutation in several heritable human disorders. These repeats are usually GC rich (e.g., over 65% GC) and are highly polymorphic in the normal population. Fragile X syndrome and myotonic dystrophy (DM) are examples of diseases in which premutation alleles cause little or no disease in the individual, but give rise to significantly amplified repeats in affected progeny. This newly identified mechanism of penetration has so far been identified in the following diseases: Fragile X syndrome (FRAXA); spinal and bulbar muscular atrophy (SMBA); myotonic dystrophy (DM); Huntington""s disease (HD); spinocerebrellar ataxia type 1 (SCA1) fragile XE (E site) mental retardation (FRAXE-MR) and dentatorubral pallidoluysian atrophy (DRAPLA). Triplet repeats are found both near to, and within additional genes, as one can learn from screening data bases of gene sequences. It is probable that in the future it will be found that the same penetration mechanism is responsible for the existence of additional genetic diseases.
Of the seven diseases listed hereinabove, Fragile XA is the most common. It is a recessive X-linked genetic disorder (therefore affecting mostly males) with an incomplete penetrance. The syndrome is difficult to diagnose in newborns and the disease features accumulate slowly with age. Developmental delay and mental retardation are the predominant clinical features of Fragile X syndrome. Mental retardation varies from extreme to borderline with the average IQ in the moderately retarded range. Female patients are more mildly affected, with few somatic signs and generally the retardation falls in to the mildxe2x80x94borderline category. Fragile-XA is one of the most common forms of mental retardation. It is the most common cause (one in 1500 males and one in 2500 females) of mental retardation from a single gene defect. It is also one of the commonest heritable disorders and the most common familial, heritable mental retardation. Furthermore, as the Fragile XA disease appears in all ethnic groups studied so far, it may be considered one of the most common single-gene disorders found in humans.
Fragile XA is characterized by an incomplete penetrance, consequently (i) some males are normal transmitting males"" (NTMs); They are clinically normal, but their positions in the genetic pedigree makes them obligate carriers of the mutated allele. (ii) About a third of the carrier women (heterozygotes) exhibit slight symptoms of mental disturbances. The gene responsible for the syndrome was located to chromosomal position Xq27.3. Once it was cloned, it was found that within the gene there is a (CGG)n repeat that is highly polymorphic in the number (n) of repeats. This sequence is located in the 5xe2x80x2 non-translated region of the gene. A survey conducted among healthy individuals and among individuals who suffer from the syndrome has shown that the number of triplet repeats of the sequence CGG in said polymorphic locus in the first group (normal individuals) is lower than the number of such repeats in the second group (that suffers from the syndrome). While the number of CGG repeats that characterizes X chromosomes derived from healthy individuals is low, e.g., 6-52; the number of repeats in carriers is medium, e.g., 50-200; and the number of triplet repeats in individuals who suffer from the syndrome is high, e.g., 230-1000.
It was also found that when the number of CGG repeats in the FMR1 gene increases over 230, the DNA in the 5xe2x80x2 region of the gene is characterized by an abnormal number of methylated cytosine residues. This methylation covers also the promoter region of the gene and therefore causes its failure to replicate and the lack of expression of the FMR1 protein. This lack of expression and the changes in structure and organization of the DNA are most probably the direct molecular cause for the phenotype associated with the fragile XA syndrome (Caskey et al., Science, 1992, 256(5058):784-9; Pieretti et al., Cell, 1991, 66(4):817-22; and Annemieke, Cell, 1991, 65:905-914.
NTMs carry numbers of CGG repeats outside the range of normal and below those found in affected males. Such males transmit the repeats to their progeny with relatively small changes in the number of repeats. On the other hand, females who carry similar premutation alleles are prone to bear progeny (male or female) with large expansion of the repeats region. Thus, large CGG amplification associated with fragile XA syndrome appears to be predominantly a female meiotic event. See, Caskey et al. Science, 1992, 256:784-789.
Many fragile XA diseased individuals were found to be mosaic with respect to the number of the CGG trinucleotide repeats characterizing different cells in their body, a phenomenon indicating somatic instability of expanded repeats.
Instability, characterized by expansion of trinucleotide repeats, is observed also in DM, HD, FRAXE, DRPLA and SCA1 pedigrees. As opposed to FRAXA, DM and FRAXE high risk alleles can expand to similar extent through both male and female meioses and, to the best of our knowledge, somatic mosaicism has not yet been observed in DM and FRAXE patients. High risk alleles have yet to be found for HD and DRPLA, that is, alleles of these diseases either cause or do not cause the disease. Nevertheless, HD repeats are also unstable in more than 80% of meiotic transmissions; on the other hand, they are characterized by increasing, or alternatively, decreasing number of repeats with the largest increase occurring in paternal transmission (Duyao. et al. Nature Genetics, 1993, 4:387-392), whereas DRPLA alleles have a tendency to increase in size along generations. See, Nagafuchi et al. Nature Genetics, 1994, 6:14-18; Koide et al. Nature Genetics, 1994, 6:9-13.
Attempts to correlate the size of trinucleotide repeat mutations and the severity of the associated genetic diseases were made for Fragile XA syndrome, Myotonic Dystrophy, Dentatorubral Pallidoluysian Atrophy and Spinocerebellar Ataxia Type 1.
For Fragile XA, as expected, median IQ score was significantly lower for females carrying a fully expanded mutation (above 230 repeats) than for females carrying a premutation (50-200 repeats) on one of their X chromosomes. On the other hand, no significant relationship was found between IQ score and number of CGG repeats, see, Taylor et al. JAMA, 1994, 271:507-514. Nevertheless, it was found that prenatal DNA studies of the number of trinucleotide repeats characterizing Myotonic Dystrophy alleles can improve the estimation of clinical severity; and that the number of CAG trinucleotide repeats in Spinocerebellar Ataxia Type 1 and Dentatorubral Pallidoluysian atrophy is correlated with increased progression of the disease (Nagafuchi et al. Nature Genetics, 1994, 6:14-18; Koide et al. Nature Genetics, 1994, 6:9-13; Orr et al. Nature Genetics, 1993, 4:221-226).
Attempts to correlate between the size of trinucleotide repeat mutations and the age of onset of Huntington""s Disease resulted in finding a reverse correlation confined to the upper range of trinucleotide repeat numbers (ca. 60-100 repeats), see Andrew S. E. et al. (1993) Nature Genetics, 4:398-403.
Furthermore, for Spinocerebellar Ataxia Type 1 and Dentatorubral Pallidoluysian Atrophy (Nagafuchi S. et al. (1994) Nature Genetics, 6:14-18; Koide R. et al. (1994) Nature Genetics, 6:9-13), a direct correlation between the number of the (CAG)n trinucleotide repeats expansion and earlier ages of onset was found.
Amplification of GC-rich Sequences
In most cases triple repeats are GC-rich sequences, containing 65-100% G or C nucleotides in each strand. Other sequences of genes and other regions in genomes of various organisms are also known to include high G and C nucleotides.
As used herein in the specification and in the claims section below, a GC-rich sequence is defined to include above 50%, between 50% and 60%, above 60%, between 60% and 70%, above 70%, between 70% and 80%, above 80%, between 80% and 90%, above 90%, between 90% and 100%, or 100% G or C nucleotides in both strands. The length of such a sequence can range from 3 base pairs to 50,000 base pairs or more. In many cases, the length of such a sequence is between tens (10-99) of base pairs to hundreds (100-999) or thousands (1000-9,999) of base pairs.
It is known to be difficult to amplify GC rich DNA sequences using conventional amplification conditions, such as conventional polymerase chain reaction (PCR) using Thermophilus aquaticus (Taq) DNA polymerase or equivalents. Several prior art methods have already been suggested and are presented herein.
Larsen et al. (Hum Genet, 1997, 100(5-6):564-8) teaches a method a method for analysis of the FRAXA (CGG)n region in the normal and premutation range. The method is based on polymerase chain reaction (PCR) amplification of DNA extracted from whole blood or eluted from dried blood spots on filter paper, followed by automated capillary electrophoresis and detection by multicolour fluorescence. As indicated by the authors, this method suffers severe limitations. First, it is not at all effective in amplifying full-mutation alleles. Second, due to the capillary electrophoresis procedure, it is cumbersome and time consuming.
Passadore et al. (Biochem J, 1995, 308(Pt 2):513-9) teach the use of distamycin and five distamycin analogs in polymerase-chain reaction (PCR). It is shown that the use of such nucleotide analogs improves yields in some but not all cases.
Similarly, U.S. Pat. No. 5,658,764 to Pergolizzi et al. teaches a method for amplifying and detecting specific GC-rich nucleic acid sequences contained in a nucleic acid or in a mixture of nucleic acids, which includes treating a separate nucleic acid containing the specific sequence with a molar excess of primers and a polymerase and extending the primers in the presence of dATP, dCTP, TTP, and an analogue of dGTP, such as 7-Deaza-2xe2x80x2 deoxyguanosine triphosphate. The use of a dGTP analogue is limiting because one needs to use a highly thermostable DNA polymerase such as Pfu, which is cost ineffective, and the size of the amplified fragment is relatively restricted, making this method not useful for example, for amplification of pre-mutated or mutated alleles of the FMR1 gene which cover from 600 bp up to 6 kb).
Still similarly, Turner et al. (Biotechniques, 1995, 19(1):48-52) teaches the use of deoxyinosine in PCR to improve amplification of GC-rich DNA.
Yet similarly, Nakahara et al. (Nucleic Acids Res, 1998, 26(7):1854-1856) teach that inosine 5xe2x80x2-triphosphate increases the yield of nucleic acid sequence-based amplification (NASBA) products targeting GC-rich and intramolecular base-paired viroid RNA.
Condorelli et al. (Clin Genet, 1996, 50(5):366-371) teaches that amplification across CGG repeats can be inefficient and unreliable due to their 100% G+C base composition and the use of the exonuclease-deficient Pfu polymerase for amplification and detection of the CGG repeats at the FRAXA. Pfu, however, is known to be cost-ineffective. In addition, in many cases, the use of improved MetaPhor gel electrophoretic separation was required to detect amplification bands over smears.
Guldberg et al. (Nucleic Acids Research, 1998, 26(6):1548-1549) teaches the detection of mutations in GC-rich DNA by bisulfite denaturing gradient gel electrophoresis (DGGE) in combination with PCR and xe2x80x98GC-clampingxe2x80x99. DGGE, however, is cumbersome, requires high calibration and highly skilled personnel for operation and is therefore limiting.
Schuchard et al. (Biotechniques, 1993, 14(3):390-394) teaches a two-step cycle PCR method, termed xe2x80x9chot PCR, for amplification of GC-rich DNA sequences. Using this method short sequences containing about 75% G+C were amplifyable. The two-step cycle that has been developed employs a 94xc2x0 C. denaturation step and an annealing-elongation step between 70xc2x0 C. and 80xc2x0 C., with or without formamide. This method fails to efficiently amplify sequences having higher GC content.
Henke et al. (Nucleic Acids Res, 1997, 25(19):3957-3958) teaches the use of betaine to improve the PCR amplification of GC-rich DNA sequences by reducing the formation of secondary structure caused by GC-rich regions.
Culjkovic et al. (Brain Res Brain Res Protoc, 1997, 2(1):44-46) teach that circumventing a GC rich region present close to the HD gene by primer selection improves PCR amplification of the CAG trinucleotide repeats thereof.
Sequencing of GC-rich Sequences
Similar difficulties and similar methods were developed over the years for the sequencing of GC rich sequences by conventional dideoxy nucleotides based sequencing reactions. Thus, nucleotide analogs producing lower melting temperatures and reagents that either lower the melting temperature and/or stabilize single stranded DNA as such were employed in sequencing reactions in an attempt to sequence GC rich sequences.
Gel Electrophoresis-free DNA Sequencing
WO9813523A1 to Nyren, Ronaghi et al. (Science, 1998, 281(5375):363, 365, Nyren et al. (Anal Biochem, 1997, 244(2):367-373) Ronaghi et al. (Anal Biochem, 1996, 242(1):84-9) and Nyren et al. (Anal Biochem, 1993, 208(1):171-175), all of which are incorporated by reference as if fully set forth herein, teach a method of identifying a base at a target position in a single-stranded sample DNA sequence wherein an extension primer, which hybridizes to the sample DNA immediately adjacent to the target position is provided and the sample DNA and extension primer are subjected to a polymerase reaction in the presence of a deoxynucleotide or dideoxynucleotide, whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, any release of PPi being detected enzymatically, different deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incorporated, characterized in that, the PPi-detection enzyme(s) are included in the polymerase reaction step and in that in place of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for said PPi-detection enzyme. The method advantageously permits large-scale non-electrophoretic solid phase DNA sequencing, which allows for continuous determination of the progress of the polymerization reaction with time.
Thus, an approach for real-time DNA sequencing without the need for electrophoresis has been developed. The approach relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (Nyren, Anal Biochem, 1987, 167:235-238). The PPi formed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the sequencing procedure, immobilized single-stranded template is used in a repeated cycle of deoxynucleotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary bases were incorporated. An increased signal-to-noise ratio was obtained by substitution of deoxyadenosine alpha-thiotriphosphate (dATP alpha S) for the natural deoxyadenosine triphosphate, dATP alpha S is efficiently used by the DNA polymerase, but is not recognized by the luciferase. The possibility for parallel processing of many samples in an automated manner is discussed in the above recitations.
PCT/U.S.90/06178 (WO 91/06678), which is incorporated by reference as if fully set forth herein, teaches an instrument and method to determine the nucleotide sequence in a DNA molecule without the use of a gel electrophoresis step. The method uses an unknown primed single stranded DNA sequence which is immobilized or entrapped within a chamber with a polymerase so that the sequentially formed DNA can be monitored at each addition of blocked nucleotide by measurement of the presence of an innocuous marker on specified deoxyribonucleotides or deoxynucleotides.
PCT/U.S.92/07678 (WO 93/05183), which is incorporated by reference as if fully set forth herein, teaches a multistep base addition sequencing scheme (BASS) for the rapid sequencing of oligonucleotides (DNA and RNA) involving the steps of attaching a plurality of DNA or RNA strands to be sequenced to a coated support, enzymatically adding a modified nucleotide including a blocking group and a reporter group to the strands, detecting the modified nucleotide via the reporter group, removing the blocking group of the modified nucleotide and repeating these steps until the RNA or DNA is sequenced.
U.S. Pat. No. 5,650,277, which is incorporated by reference as if fully set forth herein, teaches a method aimed at the quantification of di- and trinucleotide repeats in a nucleic acid of interest by (a) if the nucleic acids are not already single stranded, treating a sample containing the nucleic acids of interest to obtain unpaired nucleotide bases spanning the position of the repeats and flanking regions; (b) contacting the unpaired nucleotide bases with an oligonucleotide primer capable of hybridizing with a stretch of nucleotide bases present in the nucleic acid of interest 3xe2x80x2 of the trinucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest; (c) ensuring that the examined nucleic acid and the oligonucleotide primer are confined to a reaction chamber at all further steps; (d) contacting the duplex with a primer extension unit which is capable of base pairing with the first nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (e) eliminating non-incorporated primer extension units; (f) contacting the template primer duplex with a primer extension unit which is capable of base pairing with the second nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (g) eliminating non-incorporated primer extension units; (h) contacting the template primer hybrid with a primer extension unit which is capable of base pairing with the third nucleotide base in the core sequence of the repeats; a detection moiety containing, primer extension unit which is capable of base pairing with a nucleotide base 5xe2x80x2 of the repeats region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the trinucleotide repeats; and a template dependent extension enzyme; (i) eliminating non-incorporated primer extension units; (j) detecting for the presence of detection moiety containing primer extension unit; (k) repeating steps (d) to (j) until detecting the detection moiety. The number of repeats as stated under (k) enables the determination of the number of trinucleotide repeats, therefore enabling determination of the exact repetition number.
However, all of the above methods are highly ineffective or inoperative when sequencing of GC rich sequences, especially sequences containing 100% G+C nucleotides, is attempted due to the formation of stable secondary structures in the nucleic acid being sequenced which hamper the sequential incorporation of nucleotides to a growing chain duplexed thereto.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods and kits for characterizing GC rich nucleic acid sequences devoid of the above limitations.
According to one aspect of the present invention there is provided a method of characterizing a GC rich region of a nucleic acid of interest comprising the steps of (a) contacting the nucleic acid of interest with an agent that modifies cytosine or guanine residues into residues complementary to adenine or thymine for obtaining a modified nucleic acid in which the cytosine or guanine residues are replaced by the residues complementary to adenine or thymine; (b) amplifying the modified nucleic acid by amplification primers being hybridizable with the modified nucleic acid and being designed for directing exponential amplification of at least a portion of the modified nucleic acid, for obtaining an amplification product corresponding to the GC rich region; and (c) determining the size of the amplification product, thereby characterizing the GC rich region of the nucleic acid of interest.
According to another aspect of the present invention there is provided a kit useful for characterizing a GC rich region of a nucleic acid of interest comprising carrier being compartmentalized to receive in close confinement therein one or more containers comprising a first container containing an agent effective in modifying cytosine or guanine residues of the nucleic acid of interest into residues complementary to adenine or thymine for obtaining a modified nucleic acid in which the cytosine or guanine residues are replaced by the residues complementary to adenine or thymine, a second container or containers containing amplification primers for amplifying the modified nucleic acid the primers being hybridizable with the modified nucleic acid and being designed for directing exponential amplification of at least a portion of the modified nucleic acid, for obtaining an amplification product corresponding to the GC rich region.
According to yet another aspect of the present invention there is provided a method of characterizing a GC rich region of a nucleic acid of interest comprising the steps of (a) contacting the nucleic acid of interest with an agent that modifies cytosine or guanine residues into residues complementary to adenine or thymine for obtaining a modified nucleic acid in which the cytosine or guanine residues are replaced by the residues complementary to adenine or thymine; (b) contacting the modified nucleic acid in a single stranded form with a sequencing primer hybridizeable with a stretch of nucleotides of the single stranded form of the modified nucleic acid; (c) synthesizing a complementary nucleic acid being complementary to the single stranded form of the modified nucleic acid, the synthesizing being carried out in a stepwise serial manner in which the identity of each nucleotide incorporated into the complementary nucleic acid is determined subsequent to its incorporation; and (d) determining a sequence of the single stranded form of the modified nucleic acid, thereby characterizing the GC rich region of the nucleic acid of interest.
According to further features in preferred embodiments of the invention described below, the method further comprising the step of (e) prior to step (b), amplifying the modified nucleic acid by amplification primers being hybridizable with the modified nucleic acid and being designed for directing exponential amplification of at least a portion of the modified nucleic acid, for obtaining an amplification product corresponding to the GC rich region, and using a single stranded form of the amplification product for synthesizing the complementary nucleic acid.
According to still further features in the described preferred embodiments step (c) is effected in four confinements each corresponding to one type of the four nucleotide types present in DNA.
According to still further features in the described preferred embodiments step (c) is effected in a single confinement.
According to still further features in the described preferred embodiments each the nucleotide incorporated into the complementary nucleic acid contains a removable blocking group at its 3xe2x80x2xe2x80x94OH position.
According to still further features in the described preferred embodiments each the nucleotide incorporated into the complementary nucleic acid contains a reporter group.
According to still further features in the described preferred embodiments the reporter group is removable.
According to still further features in the described preferred embodiments the reporter group is selected from the group consisting of radiolabel, fluorolabel, metal ions antibodies and chemiluminesence compounds.
According to still further features in the described preferred embodiments the identity of each nucleotide incorporated into the complementary nucleic acid is determined subsequent to its incorporation by monitoring a release of a PPi group.
According to still further features in the described preferred embodiments monitoring the release of the PPi group is effected by a PPi-detection enzyme.
According to still further features in the described preferred embodiments the method is effective in quantifying the number of trinucleotide repeats having a known core sequence and therefore a known modified core sequence in the nucleic acid of interest, wherein step (c) is effected by the cycled steps of (i) providing a first primer extension unit for base pairing with a first nucleotide base in the known modified core sequence and with a template dependent extension enzyme; (ii) eliminating non-incorporated units of the first primer extension units; (iii) providing a second primer extension unit for base pairing with a second nucleotide base in the known modified core sequence, the second nucleotide base being located adjacent to and immediately 5xe2x80x2 of the nucleotide base employed under step (i), and with a template dependent extension enzyme; (iv) eliminating non-incorporated units of the second primer extension units; (v) providing: (a) a third primer extension unit for base pairing with a third nucleotide base in the known modified core sequence, the third nucleotide base being located adjacent to and immediately 5xe2x80x2 of the nucleotide base under step (iii); (b) a reporter moiety which is conjugated with a fourth primer extension unit for base pairing with a nucleotide base 5xe2x80x2 of the repeats, the nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the modified core sequence of the trinucleotide repeats, the reporter moiety which is conjugated with the fourth primer extension unit may be present in selected cycles of this stage; and (c) a template dependent extension enzyme; (vi) eliminating non-incorporated units of the third and fourth primer extension units; (vii) if step (v) included the reporter moiety which is conjugated with the fourth primer extension unit, detecting the presence of the reporter moiety; and if no detection is obtained, (viii) repeating steps (i) to (vii) until the reporter moiety is detected, the detection of the reporter moiety being indicative of the number of trinucleotide repeats included in the nucleic acid of interest.
According to further features in preferred embodiments of the invention described below, the kit further comprising a third container containing an additional agent effective in demethylating the GC rich region of the nucleic acid of interest.
According to still further features in the described preferred embodiments the demethylating agent includes a cell lysate, an enzyme or a ribozyme for effecting enzymatic demethylation of the nucleic acid of interest.
According to still further features in the described preferred embodiments the demethylating agent includes a demethylation chemical, for effecting chemical demethylation of the nucleic acid of interest.
According to still further features in the described preferred embodiments each of the amplification primers is GC clamp-free.
According to still further features in the described preferred embodiments the modifying agent is effective in modifying cytosine residues (either methylated or unmethylated) into residues complementary to adenine.
According to still further features in the described preferred embodiments the modifying agent is effective in modifying cytosine residues (either methylated or unmethylated) into residues complementary to thymine.
According to still further features in the described preferred embodiments the modifying agent is effective in modifying guanine residues into residues complementary to adenine.
According to still further features in the described preferred embodiments the modifying agent is effective in modifying guanine into residues complementary to thymine.
According to still further features in the described preferred embodiments the modifying agent is bisulfite which modifies unmethylated cytosine residues into uracil residues.
According to still further features in the described preferred embodiments the kit further comprising a DNA polymerase, preferably, a heat stable DNA polymerase.
According to still further features in the described preferred embodiments the heat stable DNA polymerase is derived from a species selected from the group consisting of Therinophilus aquaticus, Thermus thermophilus, Pyrococcus furiosus, Thermus flavus, Bacillus stearothermophilus, Thermococcus litoralis and Escherichia coli. 
According to still further features in the described preferred embodiments the heat stable DNA polymerase is exonuclease-deficient.
According to still further features in the described preferred embodiments the primers are designed to hybridize 5xe2x80x2 to and 3xe2x80x2 to a trinucleotide repeat region in the nucleic acid of interest.
According to still further features in the described preferred embodiments trinucleotide repeat region is of a gene associated with a hereditary disease selected from the group consisting of Fragile XA syndrome (FRAXA), spinal and bulbar muscular atrophy (SMBA), myotonic dystrophy (DM), Huntington""s disease (HD), spinocerebrellar ataxia type 1 (SCA1), fragile XE mental retardation (FRAXE-MR) and dentatorubral pallidoluysian atrophy (DRAPLA).
The present invention successfully addresses the shortcomings of the presently known configurations by providing methods and kits for characterizing GC rich nucleic acid sequences, such as trinucleotide repeats in cases of, for example, fragile XA (FMR1).